Utility of a test for chromosomal malsegregation in Saccharomyces cerevisiae strain D61.M for the detection of antianeugens: test of the model combination of chlorophyllin and nocodazole.

Despite the fact that aneuploidy is a major genetic cause of human morbidity and mortality, antimutagenicity studies have used predominantly short-term tests that detect gene mutations, chromosomal aberrations, and micronuclei. Therefore, the major deficiency in the use of short-term tests for antimutagenicity studies is those that detect chromosomal malsegregation leading to aneuploidy. Thus, we initiated a study on the utility of short-term tests for the detection of antianeugenic activity. We selected strain D61.M of Saccharomyces cerevisiae, nocodazole, and chlorophyllin as a model short-term test, aneugen, and antimutagen, respectively, for our initial study. Chlorophyllin strongly inhibited the aneugenic activity of nocodazole, but had no effect on the endpoints when tested alone, in strain D61.M. To our knowledge, this is the first report of an antianeugen. Furthermore, we conclude that strain D61.M can be used as a relatively simple, inexpensive, and rapid short-term test for the study of antianeugenicity.

Induction of chromosome loss in Saccharomyces cerevisiae strain D61.M by selected benzimidazole compounds.

Twenty-two benzimidazole compounds were tested for induction of chromosome loss (CHRL) in the diploid yeast Saccharomyces cerevisiae strain D61.M. Six compounds tested positive for CHRL induction: mebendazole, albendazole, RS-9237-000, fenbendazole, 2-benzimidazolylacetonitrile, and thiabendazole. Mebendazole, albendazole, RS-9237-000, and fenbendazole were strongly positive only after modified testing media were used to enhance solubility. The compounds that tested negative for CHRL were 2-phenylbenzimidazole, 2-(2-pyridyl)benzimidazole, benzimidazole, 2-aminobenzimidazole, 2-amino-5,6-dimethylbenzimidazole, 2-(aminomethyl)benzimidazole dihydrochloride hydrate, 5,6-dimethylbenzimidazole, 2-guanidinobenzimidazole, 2-methylbenzimidazole, 2-(methylmercapto) benzimidazole, 1-methyl-2-phenylbenzimidazole, 2-benzimidazolylurea, RS-65255-000, oxibendazole, and RS-95005-000. One chemical, cambendazole, tested negative or only marginally positive. Modified testing medium was also used to enhance the solubility of 2-phenylbenzimidazole, oxibendazole, and RS-95005-000. Because no toxicity was observed with oxibendazole or RS-95005-000, the negative results obtained with these two compounds could not be considered definitive.

Induction of chromosome loss in yeast by combined treatment with neurotoxic hexacarbons and monoketones.

The neurotoxic hexacarbon compounds n-hexane, 2-hexanone and 2,5-hexanedione were tested in combination with acetone and methyl ethyl ketone for the potential to induce chromosome loss in strain D61.M of Saccharomyces cerevisiae. n-Hexane and 2-hexanone, alone or in combination, induced only marginally positive chromosome loss, whereas the metabolite and presumed proximal genetically active agent 2,5-hexanedione was strongly positive when tested alone and in combination. These observations are discussed in relation to the reported potentiation of the neurotoxic effects of these hexacarbons when exposure results from combinations with other solvents, e.g., acetone and methyl ethyl ketone. Treatments that result in neurotoxicity in experimental animals and humans and those that result in chromosome loss in a yeast genetic test system may be correlated by their activity on a common intracellular target.

Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae.

Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined. In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M. In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M. The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M. Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared. Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid. These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid. Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid. Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype. Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains. Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.

High levels of chromosome instability in polyploids of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was used to study the genetic consequences of polyploidy in a unicellular organism. Isogenic diploid (2N), triploid (3N) and tetraploid (4N) strains with a genetically marked chromosome VII (cyh2-leu1-CEN7-ade6) were constructed and were used to follow the loss of one, two or three chromosome VII's during mitosis. We found that as ploidy increased, the frequency of loss of a single chromosome VII increased: Loss of one copy of chromosome VII occurred at a rate nearly 30-fold higher in triploids and approximately 1000-fold higher in tetraploids than in the diploid. Loss of two or three copies occurred at an even greater frequency. These findings suggest either that aneuploidy (3N-1, 3N-2, 4N-1, 4N-2, 4N-3) increases genome instability or that multiple chromosome loss events occur at high frequency. Polyploidy appears to dramatically increase chromosome loss, presumably due to the inability of the cell to undergo proper chromosome segregation. The biological significance and possible causes for the instability of polyploidy in unicellular organisms such as yeast are discussed.

Observations on chromosome loss detection by multiple recessive marker expression in strain D61.M of Saccharomyces cerevisiae.

Since chromosomes of fungi are difficult to observe directly, strains have been developed in which chromosome loss can be detected by the use of genetic markers. In the diploid D61.M strain of Saccharomyces cerevisiae, the loss of a copy of chromosome VII that carries 3 dominant wild-type alleles is measured by expression of 3 recessive mutant alleles carried on the other remaining copy of chromosome VII. We have tested the hypothesis that expression of the 3 recessive alleles might be due to 3 simultaneous independent genetic events other than chromosome loss, such as mutation or recombination. We have measured, when possible, the frequencies of expression for each of these recessive alleles, independently and in combination one with another, under both selective and non-selective conditions. Our results show that simultaneous expression of these 3 recessive alleles is attributable to chromosome loss (greater than 98%). Similarly, at least 99% of the nocodazole-induced events are attributable to chromosome loss. In contrast, most if not all of the apparent chromosome loss induced by ethyl methanesulfonate is due to multiple events of mutation or recombination.

Investigations of aneuploidy-inducing chemical combinations in Saccharomyces cerevisiae.

For several years we have been investigating combinations of chemicals for their ability to induce aneuploidy. Earlier published results indicated that combinations of certain chemicals showed a potentiation effect while other combinations did not. We have continued to explore this phenomenon and report additional findings in this communication. Combinations of ethyl acetate and methyl ethyl ketone showed a potentiation effect as did 1-methyl-2-pyrrolidinone-nocodazole combinations. Combinations that did not show a potentiation effect were 2-pyrrolidinone-nocodazole and 1-methyl-2-pyrrolidinone-ethyl acetate. We also found that nocodazole, which is a potent inducer of aneuploidy in yeast extract-peptone-dextrose (YEPD) medium but not in synthetic complete (SC) medium, showed a potentiation effect with ethyl acetate in SC medium. This effect in SC medium is similar to that previously reported for nocodazole with ethyl acetate in YEPD medium. When nocodazole was dissolved in 1-methyl-2-pyrrolidinone as a concentrated stock solution, a potentiation effect occurred even at low concentrations of the solvent.

Aneuploidy induction in Saccharomyces cerevisiae by two solvent compounds, 1-methyl-2-pyrrolidinone and 2-pyrrolidinone.

A number of solvent compounds that were tested in Saccharomyces cerevisiae were potent inducers of aneuploidy, although they did not induce any other genetic effects. As an extention of these earlier findings, 1-methyl-2-pyrrolidinone was tested and was found to induce aneuploidy. Several structurally related compounds were also tested; 2-pyrrolidinone induced aneuploidy, but succinimide, pyrrolidine, 1-methylpyrrolidine, 1-methyl-3-pyrrolidinol, and 2-pyrrolidineethanol did not. Maleimide and its N-hydroxy, N-methyl, and N-ethyl derivatives were also negative for aneuploidy induction.

Effect of treatment medium on induction of aneuploidy by nocodazole in Saccharomyces cerevisiae.

While studying ways to improve responsiveness of Saccharomyces cerevisiae strain D61.M to agents that induce aneuploidy, we noted that nocodazole, which strongly induces aneuploidy when yeast cells are treated in yeast extract-peptone-dextrose (YEPD) medium, had no effect when a synthetic complete (SC) medium was used. Further study revealed that the presence of peptone was necessary for induction. Other aneuploidy-inducing agents, including ethyl acetate, acetone, and methyl benzimidazole-2-yl-carbamate (MBC), were equally active in either medium. Benomyl, which degrades to MBC, was less active in SC than in YEPD medium.

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae.

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.

Aneuploidy induced by nocodazole or ethyl acetate is suppressed by dimethyl sulfoxide.

Nocodazole and ethyl acetate have previously been shown to be potent inducers of aneuploidy in Saccharomyces cerevisiae. The elevation in aneuploidy frequency induced by high doses of these compounds was reduced in a dose-response manner in the presence of increasing concentrations of dimethyl sulfoxide. These results imply that compounds dissolved in dimethyl sulfoxide which either are weak inducers of aneuploidy or are of unknown potency may register as false negatives in routine screening procedures.

Aneuploidy and other genetic effects induced by hydroxyurea in Saccharomyces cerevisiae.

Hydroxyurea induces mitotic gene conversion, mitotic crossing-over, reverse mutation, respiration-deficient petite mutants and aneuploidy in growing cultures of Saccharomyces cerevisiae. Evidence is presented indicating that induction rather than selection is responsible for the increase in frequency of the genetic end points measured. Complications concerning the detection of aneuploidy in the presence of other genetic effects are described, and the need for following the complete protocol for confirmation of the aneuploids in any chemical screening program is emphasized.

The detection of chemically induced aneuploidy in Saccharomyces cerevisiae: an assessment of mitotic and meiotic systems.

Several systems have been evaluated for their ability to detect aneuploidy. Chromosome gain can be detected in mitotic haploid cells as well as meiotically derived haploid spores. Both chromosome gain and loss are detectable in mitotic diploid cells. Several chemicals have been identified that clearly induce aneuploidy in at least one or more of the systems.

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro.

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.

Acetone, methyl ethyl ketone, ethyl acetate, acetonitrile and other polar aprotic solvents are strong inducers of aneuploidy in Saccharomyces cerevisiae.

A diploid yeast strain D61.M was used to study induction of mitotic chromosomal malsegregation, mitotic recombination and point mutation. Several ketones (including acetone and methyl ethyl ketone) and some organic acid esters (including the methyl, ethyl and 2-methoxyethyl esters of acetic acid) and acetonitrile strongly induced aneuploidy but not recombination or point mutation. Only diethyl ketone induced low levels of recombination and point mutation in addition to aneuploidy. Related compounds were weak inducers of aneuploidy: methyl n-propyl ketone, the methyl esters of propionic and butyric acid, acetic acid esters of n- and iso-propanol and ethyl propionate. No mutagenicity was found with n-butyl and isoamyl acetate, ethyl formate, acetyl acetone (2,5-dipentanone) and dioxane. Methyl isopropyl ketone induced only some recombination and point mutation but no aneuploidy. Efficient induction was only observed with a treatment protocol in which growing cells were exposed to the chemicals during a growth period of 4 h at 28 degrees C followed by incubation in ice for more than 90 min, usually overnight for 16-17 h. Aneuploid cells could be detected in such cultures during a subsequent incubation at growth temperature if the chemical was still present. Detailed analysis showed that there was a high incidence of multiple events of chromosomal malsegregation. It is proposed that the mutagenic agents act directly on tubulin during growth so that labile microtubules are formed which dissociate in the cold. When cells are brought back to temperatures above the level critical for reassembly of tubulin and allowed to grow, faulty microtubules are formed.

Induction of aneuploidy by oncodazole (nocodazole), an anti-tubulin agent, and acetone.

Oncodazole (nocodazole) is a compound which interacts with yeast and bovine tubulin. We have shown that it induces aneuploidy in the yeast Saccharomyces cerevisiae at very low concentrations. In the course of a search for an appropriate solvent for oncodazole we observed that acetone also induces mitotic aneuploidy in yeast. This effect of acetone was greatly enhanced when the treatment of growing cells at 28 degrees C was interrupted by a period of holding at ice-bath temperature.

Semidominance of rad18-2 for several phenotypic characters in Saccharomyces cerevisiae.

Inbred diploid yeast strains heterozygous or homozygous for the rad18-2 allele and carrying markers for detection of mitotic recombination were constructed. The homozygous rad18-2/rad 18-2 strain was highly sensitive to killing by UV light, showed greatly elevated frequencies of spontaneous and induced mitotic recombination and was more sensitive to trimethoprim than the wild-type diploid. The heterozygous strain RAD18/rad 18-2 was intermediate in its response for these same phenotypic characters. These findings are discussed in the light of other studies in which incomplete dominance of genes involved in some aspect of DNA repair has been reported.

Activation of cycasin to a mutagen for Saccharomyces cerevisiae by rat intestinal flora.

Genetic test systems involving microorganisms and liver enzyme preparations may be insufficient to detect compounds that require breakdown by enzymes provided by the microbial flora of the intestinal tract. A method is described for providing such activation and for simultaneously testing the potential genetic activity of breakdown products in an indicator organism. Parabiotic chambers containing Saccharomyces cerevisiae genetic test organisms in one chamber were separated by a membrane filter from rat cecal organisms and test chemical contained in the other chamber. The genetic activities of cycasin breakdown products for mutation, gene conversion, and mitotic crossing-over in samples incubated aerobically are reported. Samples containing cycasin alone had a small but clearly increased frequency of genetic damage. Samples containing rat cecal organisms without cycasin showed no increase in genetic activity. Anaerobic incubation resulted in no increase in genetic activity in any of the samples.

Induction of mitotic recombination by certain hair-dye chemicals in Saccharomyces cerevisiae.

A number of procedures were used to test for the potential of 5 hair-dye chemicals, 4-nitro-o-phenylenediamine, 2-nitro-p-phenylenediamine, m-phenylenediamine 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate, to induce genetic damage in yeast strains D3 and D4 of Saccharomyces cerevisiae. Various plate-test procedures, short-term suspension assays in phosphate buffer and suspension assays with liver enzyme activation all proved to be ineffective for demonstrating genetic effects of these chemicals. Only suspension assays in which the yeast cells were treated with the test chemical under growing conditions for up to 72 h were effective in demonstrating the genetic activity of 4-nitro-o-phenylenediamine and 2,4-diaminoanisole sulfate. The implications of these results for testing of mutagens in yeast systems are discussed along with other supportive evidence from the literature.